The adult mammalian central nervous system (CNS) is a profoundly inhibitory environment for axon regeneration. This is due, in significant measure, to multiple axon regeneration inhibitors (ARI's) in the milieu of a CNS injury. These include Nogo, myelin-associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgp) and chondroitin sulfate proteoglycans (CSPG). Each ARI binds to complementary ligands on the nerve or axon surface, halting axon regeneration. Rapidly emerging knowledge of ARI's and their ligands provides previously unanticipated opportunities to block their actions and potentially enhance axon regeneration. The relative contributions of the different known ARl's in blocking axon regeneration in vivo are unknown. This R21 Exploratory/Developmental Grant application describes a highly directed, short-term program to block the known ARI's, individually and in combination, in a well characterized in vivo spinal cord injury model. The proposed studies take advantage of the latest findings regarding the molecular nature of ARI's. The results may direct future studies, in that they will provide comparative data on the effects of blocking the different inhibitory systems side by side in the same lesion model. Depending on the outcome, they may encourage future efforts to focus on multi inhibitor blocking rather than on blocking single systems. The "exploratory/developmental" aspects of the proposed approach include (i) multi-inhibitor blocking in a single lesion model; and (ii) the use of the tools of glycobiology as experimental therapeutics. Each ARI and/or its nerve cell surface ligand(s) are glycosylated. Moreover, glycosylation plays key structural and functional roles in inhibiting axon regeneration. The nerve cell ligand for Nogo, NgR, is a glycosylphosphatidylinositol (GPI)-Iinked glycoprotein, as is OMgp. MAG is a sialic acid binding protein that binds to nerve cell surface gangliosides GDla and GTlb, as well as to NgR. Finally, the sugar chains of CSPG mediate its inhibition of axon regeneration. The tools of glycobiology, in concert with novel biochemical tools, provide feasible means to block ARI's and enhance axon regeneration in vivo. We will use the following glyco-enzymes for this purpose: phosphatidylinositol-specific phospholipase C (PI-PLC), to cleave the GPI anchors of NgR and OMgp, neuraminidase to cleave GDla and GTlb, and chondroitinaseABC to cleave CSPG. The biochemical efficacy of each enzyme will be monitored immunohistochemically, and their effects on regeneration will be determined after spinal cord lesion (dorsal hemisection) in rats. ARI-blocking saccharides, peptides, and antibodies will also be used to further enhance the value of these studies. The resulting data may provide insight on the relative contributions of the different ARI's in an in vivo model, and may support evaluation of new approaches to enhance axon regeneration after CNS injury.